This invention relates to novel 2-alkanoylamino-4-nitrophenyl phosphorylcholine-hydroxide compounds, and, more particularly, to their use as sphingomyelinase-specific chromogenic artificial substrates in the diagnostic testing for Niemann-Pick disease.
In patients with the hereditary disorder known as Niemann-Pick disease, excessive quantitities of the naturally occurring lipid, sphingomyelin, accumulate in certain organs and tissues, due to a deficiency of sphingomyelinase, a component enzyme of all normal mammalian tissues which catalyzes the hydrolysis of sphingomyelin into its component parts, i.e., ceramide and phosphorylcholine. Measurement of sphingomyelinase activity in extracts of human cells or tissues is a proven procedure for the diagnosis of Niemann-Pick disease, the detection of healthy heterozygous carriers of the Niemann-Pick trait, and the prenatal diagnosis of fetuses afflicted with Niemann-Pick disease. Such determination of sphingomyelinase activity has previously required the use of radioactively labeled sphingomyelin, which is difficult to prepare, expensive, and available in only very limited quantities. Moreover, most clinical laboratories are not equipped to carry out assays with the radioactive sphingomyelin, and thus such testing has been restricted to research laboratories with radioactive counting facilities.
Due to the above-described limitations of the radioactive sphingomyelin, a more practical sphingomyelinase-specific artificial substrate for use in the diagnostic testing for Niemann-Pick disease has been sought for some time. A hypothetical artificial substrate potentially useful for this purpose, consisting of 2-alkanoylamino-4-nitrophenyl phosphorylcholine compounds, was proposed a few years ago by Dr. Roscoe O. Brady, one of the present co-inventors. Such compounds chemically and structurally resemble sphingomyelin, differing therefrom only by having an aromatic ring instead of a long aliphatic chain and a nitro group replacing the primary hydroxyl one carbon removed. Dr. Brady's proposal, first published in an article by Brady et al appearing in The American Journal of Medicine, Volume 51, October 1971, Pages 423-431, was based on the supposition that sphingomyelinase in a test preparation would catalyze the hydrolysis of the proposed substrate into phosphorylcholine and a 2-alkanoylamino-4-nitrophenol, and that the latter product, upon being alkalinized, would develop a yellow color proportional in intensity to the sphingomyelinase activity in the test preparation. The 1971 Brady et al. article indicated that the synthesis of the proposed substrate from a 2-amino-4-nitrophenol starting material, and an examination of its reliability in the diagnostic testing for Niemann-Pick disease, were at that time being undertaken.
As it subsequently turned out, however, Dr. Brady's proposed artificial substrate remained merely a hypothetical substance, and its conjectured usefulness for the determination of sphingomyelinase activity remained unsubstantiated, for quite some time following the 1971 Brady et al. article, as evidenced by several subsequently published articles authored by Dr. Brady appearing in Angew. Chem. Internat. Edit., Volume 12, No. 1, January 1973, Pages 1-11; "Lysosomes and Storage Diseases", Academic Press, Inc., New York and London (1973), Pages 439-452; and "Clinical Biochemistry Principles and Methods", Walter de Gruyter, New York and Berlin, (1974), Pages 1282-1284. All of these publications indicated that Dr. Brady's proposed artificial substrate had yet to be synthesized and examined for its reliability as an indicator of sphingomyelinase activity. The fact of the matter is that even though the 1971 Brady et al article even went so far as to suggest a starting material from which the proposed artificial substrate could be synthesized, the determination of the intermediate steps required to effect such synthesis presented a substantial amount of difficulty which led to numerous unsuccessful attempts at producing the desired end product. The complete failure that was experienced in being able in any way to effect a synthesis of Dr. Brady's proposed artificial substrate, thus left this substrate remaining as merely a hypothetical substance whose utility as a reliable chromogenic indicator of sphingomyelinase activity in the diagnostic testing for Niemann-Pick disease was still a matter of pure conjecture and incapable of being actually determined.